Combination liposomal pharmaceutical formulations

ABSTRACT

Docetaxel and doxorubicin can be formulated in liposomal pharmaceutical compositions. In various embodiments, the pharmaceutical compositions include (i) a first liposome type comprising a first lipid layer comprising an unsaturated phospholipid, cholesterol or a cholesterol derivative, DC-cholesterol, a cationic lipid, and preferably a pegylated phospholipid, and a first active pharmaceutical ingredient (API) comprising docetaxel in the first lipid layer; and (ii) a second liposome type comprising a second lipid layer, an aqueous interior, and a second API comprising doxorubicin crystallized in the aqueous interior, (iii) where the first liposome type does not comprise doxorubicin and the second liposome type does not comprise docetaxel. The pharmaceutical composition can be used to treat a subject, for example, a human subject having cancer. The cancer can be, for example, a lung cancer, preferably non-small cell lung cancer (NSCLC), colon cancer, breast cancer, or liver cancer, preferably hepatocellular carcinoma (HCC).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/127,487, filed Mar. 3, 2015, which is hereby incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention generally relates to liposomal pharmaceuticalcompositions and, in various embodiments, more specifically to liposomalpharmaceutical compositions including two different activepharmaceutical ingredients (API) in two different liposome types (e.g.,a first liposome type comprising a first API such as docetaxel, and asecond liposome type comprising a second API such as doxorubicin).

BACKGROUND

Liposome technology has been utilized for drug delivery in clinicaltherapy and scientific research. To date, a handful of liposomalpharmaceutical formulations have been approved by the US Food and DrugAdministration (“FDA”), and a number of new liposomal formulations arein clinical trials. However, the field of liposomal formulation is stillevolving and each active pharmaceutical ingredient (“API”) presentsunique challenges.

One area where liposomal formulations can be applied is in cancer APIs.For example, liposomal formulations of doxorubicin are presentlyavailable under the trade names Doxil® and Myocet®. Doxil® is apegylated (polyethylene glycol coated) liposome-encapsulated form ofdoxorubicin formerly made by Ben Venue Laboratories in the United Statesfor Janssen Products, LP, a subsidiary of Johnson & Johnson. Myocet® isa non-pegylated liposomal doxorubicin made by Enzon Pharmaceuticals forCephalon in Europe and for Sopherion Therapeutics in the United Statesand Canada. Myocet® is approved in Europe and Canada for treatment ofmetastatic breast cancer in combination with cyclophosphamide, but isnot yet approved by the FDA for use in the United States.

Despite the handful of approved liposomal pharmaceutical formulations,the field is still limited by the currently available methods of makingliposomal formulations, which present difficult problems associated withscalability and production costs. There exists a need for improvedliposomal formulations for use in drug delivery.

SUMMARY OF THE INVENTION

In various aspects and embodiments, the invention provides apharmaceutical composition including a first liposome type comprising afirst API (e.g., docetaxel) and a second liposome type comprising asecond API (e.g., doxorubicin). In various embodiments, (i) the firstliposome type comprises a first lipid layer comprising an unsaturatedphospholipid, cholesterol, and preferably a pegylated phospholipid, anda first active pharmaceutical ingredient (API) (e.g., docetaxel) in thefirst lipid layer; and (ii) a second liposome type comprising a secondlipid layer, an aqueous interior, and a second API (e.g., crystallizeddoxorubicin) in the aqueous interior. The first liposome type does notcomprise the second API (e.g., doxorubicin) and the second liposome typedoes not comprise the first API (e.g., docetaxel). The liposomes can beused to treat a subject, for example, a human subject having cancer. Thecancer can be, for example, a lung cancer, preferably non-small celllung cancer (NSCLC); colon cancer; breast cancer; or liver cancer,preferably hepatocellular carcinoma (HCC).

The invention can provide for increased efficacy and/or decreasedtoxicity, for example relative to (i) other pharmaceutical compositionswhere the first drug (e.g., docetaxel) and/or the second drug (e.g.,doxorubicin) are not in a liposomal formulation. The invention can alsoprovided superior results to known liposomal, and non-liposomal,formulation (e.g., Doxil® and Myocet®, or Taxotere®).

The invention can provide for targeted delivery of both the first andsecond API, for example to the liver. The invention can mitigateundesired side effects, for example by providing for increased drugloading, thereby reducing the amount of liposomes needed to deliver aquantity of the first drug (e.g., docetaxel) and the second drug (e.g.,doxorubicin). Also, because the second drug (e.g., doxorubicin) iscrystallized in the second liposome type, the second drug can have moretargeted delivery and less off-target side effects.

Furthermore, because the first and second API are in separate liposomes,each liposome can be designed specifically for the API it carries. Thedifferent combinations of the two liposomal formulations, includingdifferent API ratio, different dosing sequence and interval time, can beclinically identified for the treatment of different type of cancers. Italso provides physicians a flexibility to adjust the ratios of the twoliposomal APIs at patients' bedside to optimize the outcomes whileminimizing the adverse reactions for individual patients.

The invention provides a pharmaceutical composition comprising: (i) afirst liposome type comprising a first lipid layer comprising anunsaturated phospholipid, cholesterol, and preferably a pegylatedphospholipid, and a first active pharmaceutical ingredient (API)comprising docetaxel in the first lipid layer; and (ii) a secondliposome type comprising a second lipid layer, an aqueous interior, anda second API comprising doxorubicin crystallized in the aqueousinterior. The first liposome type does not comprise doxorubicin and thesecond liposome type does not comprise docetaxel.

In various embodiments, the first lipid layer and/or the second lipidlayer consist of unsaturated phospholipid and cholesterol.

In various embodiments, the first lipid layer and/or the second lipidlayer consist of unsaturated phospholipid, cholesterol, cationic lipid,and pegylated phospholipid.

In various embodiments, the first lipid layer and the second lipid layercomprise different lipid compositions (e.g., different lipid componentsand/or different amounts of certain lipid components). In variousembodiments, the first liposome and the second liposome are different(e.g., in addition to comprising different APIs, derived from differentlipid solutions and/or aqueous solutions as described below).Alternatively, in some embodiments, the first and second lipid layersare essentially the same (e.g., other than comprising different APIs,comprising essentially the same lipid components and amounts thereof).

In various embodiments, docetaxel is the only API in the first liposometype and/or doxorubicin is the only API in the second liposome type.

In various embodiments, the first lipid layer and/or the second lipidlayer comprise: about 20-75%, preferably about 30-60%, (molar)unsaturated phospholipid; about 10-60%, preferably 20-50%, (molar)cholesterol; about 5-75%, preferably about 10-60%, (molar) cationiclipid; and about 0-20%, preferably 1-10%, (molar) pegylatedphospholipid.

In various embodiments, the molar ratio of the first lipid layercomponents:doxorubicin is about 100:1 to about 2:1, preferably about20:1 to about 5:1; and the molar ratio of the second lipid layercomponents:docetaxel is about 100:1 to about 2:1, preferably about 20:1to about 5:1.

In various embodiments, the molar ratio of doxorubicin:docetaxel isabout 10:1 to 1:10, preferably about 5:1 to 1:5, and more preferablyabout 3:1 to 1:3.

In various embodiments, the unsaturated phospholipid comprises apolyunsaturated phospholipid or a monounsaturated phospholipid,preferably a phosphatidylcholine, and more preferably and soyphosphatidylcholine or 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine(DOPC).

In various embodiments, the cholesterol comprises a cholesterolderivative, preferably a cationic cholesterol derivative, morepreferably an amino cholesterol derivative, and still more preferablydimethylaminoethanecarbamoyl-cholesterol (DC-cholesterol).

In various embodiments, the pegylated phospholipid comprises aphosphoethanolamine, preferably a1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) and wherein thepegylation is a PEG 500 to PEG 3000, preferably PEG 2000.

In various embodiments, the pharmaceutical composition is formulated forintravenous administration.

In various embodiments, the Z-average particle size of the liposomes isabout 10-200 nm, preferably about 15-150 nm, and more preferably about20-120 nm.

In various embodiments, upon intravenous administration to a subject, atleast about 10% of the composition is delivered to the liver.

In various embodiments, the pharmaceutical composition is for use as amedicament.

In various embodiments, the pharmaceutical composition is for use as acancer therapeutic.

The invention also provides a pharmaceutical composition consisting of:(i) a plurality of liposomes of the first type as described or claimedherein; (ii) a plurality of liposomes of the second type as described orclaimed herein; and (iii) one or more pharmaceutical excipients.

The invention also provides a method of making the pharmaceuticalcomposition as described or claimed herein. For example, the inventionprovides a method of making a pharmaceutical composition as described orclaimed herein, comprising: (i) making a first liposome type byintroducing a first lipid solution of an unsaturated phospholipid,cholesterol, a cationic lipid, docetaxel, and preferably a pegylatedphospholipid in ethanol through a first or more port of a multi-portmanifold into a mixing chamber and introducing a aqueous solutionthrough a second or more port of the multi-port manifold into the mixingchamber, the liposome formed in the mixing chamber exit the mixingchamber through a third or more exit port, wherein the resulting firstliposome type does not comprise doxorubicin; (ii) making a secondliposome type by introducing a second lipid solution in ethanol througha first or more inlet port of a multi-port manifold into a mixingchamber and introducing an aqueous solution through a second or moreinlet port of the multi-port manifold into the mixing chamber, theliposome formed in the mixing chamber exit the mixing chamber throughone or more outlet port. The liposome is then incubated with doxorubicinto encapsulate doxorubicin, wherein the resulting second liposome typedoes not comprise docetaxel; and (iii) combining predetermined amountsof the first liposome type and the second liposome type, thereby makingthe pharmaceutical composition as described or claimed herein.

The invention provides a method of treating a subject comprisingadministering an effective amount of any of the pharmaceuticalcompositions of the invention described or claimed herein. In variousembodiments, the subject has a cancer. In various embodiments, thecancer is a lung cancer, preferably non-small cell lung cancer (NSCLC);colon cancer; breast cancer; or liver cancer, preferably hepatocellularcarcinoma (HCC).

These and other advantages of the present technology will be apparentwhen reference is made to the accompanying drawings and the followingdescription.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates volume-weighted particle size distribution ofliposomal formulation CPT307A determined by dynamic light scattering.

FIG. 2 illustrates volume-weighted particle size distribution ofliposomal formulation CPT307B determined by dynamic light scattering.

FIG. 3 illustrates volume-weighted particle size distribution ofliposomal formulation CPT319A determined by dynamic light scattering.

FIG. 4 illustrates volume-weighted particle size distribution ofliposomal formulation CPT319B determined by dynamic light scattering.

FIG. 5 presents a cryo transmission electron microscopy (TEM) image ofliposomal formulation CPT319B.

FIG. 6 presents a negative stained TEM image of liposomal formulationCPT319B.

FIG. 7 illustrates non-small cell lung cancer (NSCLC) tumor growthcurves and tumor weight inhibition percentages (TW Inh %) afteradministration with liposomal formulations (CPT319A, CPT319B, orCPT319AB) or non-liposomal formulations of docetaxel/doxorubicin,compared to the control group.

FIG. 8 illustrates NSCLC tumor growth curves and tumor weight inhibitionpercentages (TW Inh %) after administration with liposomal formulations(CPT307A, CPT307B, or CPT307AB) or non-liposomal formulations ofdocetaxel/doxorubicin, compared to the control group.

FIG. 9 illustrates colon cancer tumor growth curves after administrationwith three different doses of liposomal formulation (CPT319AB), comparedto the control group.

FIG. 10 illustrates colon cancer tumor growth curves afteradministration with three different doses of liposomal formulation(CPT307AB), compared to the control group.

FIG. 11 illustrates hepatocellular carcinoma tumor growth curves afteradministration with liposomal formulation (CPT319AB), compared to thecontrol group.

FIG. 12 illustrates NSCLC tumor growth curves after administration withliposomal formulations (CPT307A, B, or AB and CPT319A, B, or AB),compared to the control group.

FIG. 13 illustrates plasma concentration curves of doxorubicin afteradministration with liposomal formulations (CPT307A or CPT319A),compared to non-liposomal formulations of docetaxel/doxorubicin. *BLOQ:Below Limit of Quantitation.

FIG. 14 illustrates liver concentration curves of doxorubicin afteradministration with liposomal formulations (Doxil®, CPT319A, or CPT221),as well as the DC-Cholesterol percentages of each liposomal formulations(DC-chol %).

While the invention comprises embodiments in many different forms, thereare shown in the drawings and will herein be described in detail severalspecific embodiments with the understanding that the present disclosureis to be considered as an exemplification of the principles of thetechnology and is not intended to limit the invention to the embodimentsillustrated.

DETAILED DESCRIPTION

In various aspects and embodiments, the invention provides apharmaceutical composition including a first liposome type comprising afirst API (e.g., docetaxel) and a second liposome type comprising asecond API (e.g., doxorubicin). In various embodiments, (i) the firstliposome type comprises a first lipid layer comprising an unsaturatedphospholipid, cholesterol, and preferably a pegylated phospholipid, anda first active pharmaceutical ingredient (API) (e.g., docetaxel) in thefirst lipid layer; and (ii) a second liposome type comprising a secondlipid layer, an aqueous interior, and a second API (e.g., crystallizeddoxorubicin) in the aqueous interior. The first liposome type does notcomprise the second API (e.g., doxorubicin) and the second liposome typedoes not comprise the first API (e.g., docetaxel).

As used herein, the phrase “the first liposome type does not comprisethe second API and the second liposome type does not comprise the firstAPI” or “the first liposome type does not comprise doxorubicin and thesecond liposome type does not comprise docetaxel” means that firstliposome type includes essentially none, or a negligible amount, of thesecond API (e.g., doxorubicin) and the second liposome type includesessentially none, or a negligible amount, the first API (e.g.,docetaxel). As will be understood by one of ordinary skill in the art,any compound has a partition coefficient between aqueous and non-aqueousphases that are in equilibrium. For example, given a hydrophobiccompound (e.g., docetaxel) in a non-aqueous phase (e.g., lipid layer ofa liposome) that is in equilibrium with an aqueous phase (e.g., aqueousinterior of a liposome), the partition coefficient dictates that somenegligible amount (e.g., essentially none) of the hydrophobic compoundcan be found partitioned to the aqueous phase. In other words, anyamount of doxorubicin the first liposome type in the experimentalExamples below can be considered as essentially none, or a negligibleamount—i.e., the first liposome type does not comprise doxorubicin.Likewise, any amount of docetaxel the second liposome type in theexperimental Examples below can be considered as essentially none, or anegligible amount—i.e., the second liposome type does not comprisedocetaxel.

The liposomes can be used advantageously to treat a subject, forexample, a human subject having cancer. The cancer can be, for example,a lung cancer, preferably non-small cell lung cancer (NSCLC); coloncancer; breast cancer; or liver cancer, preferably hepatocellularcarcinoma (HCC). The invention can provide for increased efficacy and/ordecreased toxicity, for example relative to (i) other pharmaceuticalcompositions where the first drug (e.g., docetaxel) and/or the seconddrug (e.g., doxorubicin) are not in a liposomal formulation. Theinvention can also provided superior results to known liposomal, andnon-liposomal, formulation (e.g., Doxil® and Myocet®, or Taxotere®).

The invention can provide for targeted delivery of both the first andsecond API, for example to the liver. The invention can mitigateundesired side effects, for example to load a water insoluble drug intothe liposome to eliminate the toxic solvent used for the non-liposomaldrug (e.g., docetaxel) formulations thus to abolish the toxic solventinduced adverse react. Furthermore, because the first and second API arein separate liposomes, each liposome can be designed specifically forthe API it carries. The different combinations of the two liposomalformulations, including different API ratio, different dosing sequenceand interval time, can be clinically identified for the treatment ofdifferent type of cancers. It also provides physicians a flexibility toadjust the ratios of the two liposomal APIs at patients' bedside tooptimize the outcomes while minimizing the adverse reactions forindividual patients.

The various features of such liposomes, as well as pharmaceuticalcompositions including the liposomes and methods of using and making theliposomes are discussed, in turn, below.

Active Pharmaceutical Ingredient (API)

In various aspects and embodiments, the compositions includes a firstAPI (e.g., docetaxel in a first liposome type) and a second API (e.g.,doxorubicin in a second, separate, liposome type). While docetaxel anddoxorubicin are presented as illustrative examples, other embodimentsare possible where a different first API is in the lipid layer of thefirst liposome type and a different second API is in (e.g., crystallizedin) the aqueous interior of the second liposome type.

In various embodiments, the two liposome types can separately includetwo (or more) anticancer agents, anti-inflammatory agents, anti-diabeticagents, anti-fungal agents, and/or antibiotic agents. Where additionalAPIs (i.e., more than two) are present, the first and/or second liposometypes can include the additional API(s). Alternatively, the additionalAPIs can be included in liposome(s) separate from the first and secondliposome types (e.g., in a third liposome type).

Docetaxel (as generic or under the trade name Taxotere® or Docecad®) isa clinically well-established anti-mitotic chemotherapy medication thatworks by interfering with cell division. Docetaxel is approved by theFDA for treatment of locally advanced or metastatic breast cancer, headand neck cancer, gastric cancer, hormone-refractory prostate cancer andnon small-cell lung cancer. Docetaxel can be used as a single agent orin combination with other chemotherapeutic drugs as indicated dependingon specific cancer type and stage.

Docetaxel is a member of the taxane drug class, which also includes thechemotherapeutic medication paclitaxel. Accordingly, in someembodiments, docetaxel can be substituted for another taxane that can bedisposed within the lipid layer of the liposome.

The optimal dose scheduling of taxanes remains unconfirmed, but moststudies find significant mortality benefit following either a three-weekor a one-week administration schedule. While some research suggestsweekly administration as an optimal schedule, the official docetaxelpackage insert recommends administration every three weeks. Importanttoxicities to note include neutropenia, febrile neutropenia andneurosensory disturbances. Such toxicities have been well documented inPhase II and Phase III clinical trials and can be anticipated andsubsequently managed.

In various embodiments, the invention can increase the efficacy of,and/or decrease undesired side effects from, the docetaxel.

Doxorubicin (trade name Adriamycin®; pegylated liposomal form trade nameDoxil®; nonpegylated liposomal form trade name Myocet®), also known ashydroxydaunorubicin and hydroxydaunomycin, is a drug used in cancerchemotherapy and derived by chemical semisynthesis from a bacterialspecies. It is an anthracycline antibiotic (note: in this context, thisdoes not mean it is used to treat bacterial infections) closely relatedto the natural product daunomycin and like all anthracyclines, it isbelieved to work by intercalating DNA, with the most serious adverseeffect being life-threatening heart damage. It is commonly used in thetreatment of a wide range of cancers, including hematologicalmalignancies (blood cancers, like leukaemia and lymphoma), many types ofcarcinoma (solid tumors) and soft tissue sarcomas. It is often used incombination chemotherapy as a component of various chemotherapyregimens. In some embodiments, doxorubicin can be substituted foranother anthracycline or another anticancer agent that can be disposedwithin the aqueous interior of the liposome.

Common adverse effects of doxorubicin include hair loss (seen in most ofthose treated with the drug), myelosuppression (a compromised ability ofthe body's bone marrow to produce new blood cells), nausea and vomiting(which are seen in roughly 30-90% of people treated with the drug), oralmucositis, oesophagitis, diarrhea, skin reactions (including hand-footsyndrome) and localized swelling and redness along the vein in which thedrug is delivered. Less common, yet serious reactions includehypersensitivity reactions (including anaphylaxis), radiation recall,heart damage and liver dysfunction.

The drug is administered intravenously, as the hydrochloride salt. It issold under a number of different brand names, including Adriamycin® PFS,Adriamycin® RDF, or Rubex®. Doxorubicin is photosensitive, andcontainers are often covered by an aluminum bag and/or brown wax paperto prevent light from affecting it. Doxorubicin is also available inliposome-encapsulated forms as Doxil® (pegylated form), Myocet®(nonpegylated form), and Caelyx®, although these forms must also begiven by intravenous injection.

In various embodiments, the invention can increase the efficacy ofand/or decrease undesired side effects from, the doxorubicin.

In some embodiments, the API may be a polynucleotide (including anoligonucleotide) a protein or a small molecule.

In one embodiment the API is a polynucleotide. The polynucleotide may bea genomic DNA fragment, cDNA, mRNA, ssRNA, dsRNA, microRNA, siRNA,shRNA, sdRNA, DsiRNA, LNA, and antisense DNA or RNA.

Alternatively, the API may be a small molecule drug. Preferably, themolecule has a molecular weight from about 1500 g/mole to about 50g/mole.

An API can include, for example, two or more of the following: ananticancer agent, an antibiotic agent, an antiviral agent, ananti-fungal agent, or an analgesic.

Exemplary anticancer agents may include but are not limited acivicin,aclarubicin, acodazole, ametantrone, aminoglutethimide, anthramycin,asparaginase, azacitidine, azetepa, bisantrene, bleomycin, busulfan,cactinomycin, calusterone, caracemide, carboplatin, carfilzomib,carmustine, carubicin, chlorambucil, cisplatin, cyclophosphamide,cytarabine, dacarbazine, dactinomycin, daunorubicin, dezaguanine,diaziquone, docetaxel, doxorubicin, epipropidine, erlotinib, etoposide,etoprine, floxuridine, fludarabine, fluorouracil, fluorocitabine,hydroxyurea, iproplatin, leuprolide acetate, lomustine, mechlorethamine,megestrol acetate, melengestrol acetate, mercaptopurine, methotrexate,metoprine, mitocromin, mitogillin, mitomycin, mitosper, mitoxantrone,mycophenolic acid, nocodazole, nogalamycin, oxisuran, paclitaxel,peliomycin, pentamustine, porfiromycin, prednimustine, procarbazinehydrochloride, puromycin, pyrazofurin, riboprine, semustine,sparsomycin, spirogermanium, spiromustine, spiroplatin, streptozocin,talisomycin, tegafur, teniposide, teroxirone, thiamiprine, thioguanine,tiazofurin, triciribine phosphate, triethylenemelamine, trimetrexate,uracil mustard, uredepa, vinblastine, vincristine, vindesine,vinepidine, vinrosidine, vinzolidine, zinostatin and zorubicin.

In specific embodiments, the anti-cancer agent is chosen fromcarfilzomib, daunorubicin, doxorubicin, paclitaxel, docetaxel,cisplatin, carboplatin, cytarabine, floxuridine, fludarabine,fluorouracil, iproplatin, leuprolide acetate, and methotrexate.

Exemplary antibiotic agents may include but are not limited toaminoglycoside; amikacin; gentamicin; kanamycin; neomycin; netilmicin;steptomycin; tobramycin; ansamycins; geldanamycin; herbimycin;carbacephem; loracarbef; carbacepenem; ertapenem; doripenem;imipenem/cilastatin; meropenem; cephalosporin; cefadroxil; cefazolin;cefalotin or cefalothin; cefalexin; cefaclor; cefamandole; cefoxitin;cefprozil; cefuroxime; cefixime; cefdinir; cefditoren; cefoperazone;cefotaxime; cefpodoxime; ceftazidime; ceftibuten; ceftizoxime;ceftriaxone; cefepime; ceftobiprole; glycopeptide; teicoplanin;vancomycin; macrolides; azithromycin; clarithromycin; dirithromycin;erythromicin; roxithromycin; troleandomycin; telithromycin;spectinomycin; monobactam; aztreonam; penicillins; amoxicillin;ampicillin; azlocillin; carbenicillin; cloxacillin; dicloxacillin;flucloxacillin; mezlocillin; meticillin; nafcillin; oxacillin;penicillin, piperacillin, ticarcillin; bacitracin; colistin; polymyxinB; quinolone; ciprofloxacin; enoxacin; gatifloxacin; levofloxacin;lomefloxacin; moxifloxacin; norfloxacin; ofloxacin; trovafloxacin;sulfonamide; mafenide; prontosil (archaic); sulfacetamide;sulfamethizole; sufanilimide (archaic); sulfasalazine; sulfisoxazole;trimethoprim; trimethoprim-sulfamethoxazole (co-trimoxazole) (TMP-SMX);tetracycline; demeclocycline; doxycycline; minocycline; oxytetracycline;tetracycline; arsphenamine; chloramphenicol; clindamycin; lincomycin;ethambutol; fosfomycin; fusidic acid; furazolidone; isoniazid;linezolid; metronidazole; mupirocin; nitrofuantoin; platensimycin;polymyxin, purazinamide; quinupristin/dalfopristin; rifampin orrifampicin; and timidazole.

Exemplary antiviral agents may include, but are not limited tothiosemicarbazone; metisazone; nucleoside and/or nucleotide; acyclovir;idoxuridine; vidarabine; ribavirin; ganciclovir; famciclovir;valaciclovir; cidofovir; penciclovir; valganciclovir; brivudine;ribavirin, cyclic amines; rimantadine; tromantadine; phosphonic acidderivative; foscamet; fosfonet; protease inhibitor; saquinavir;indinavir; ritonavir; nelfinavir; amprenavir; lopinavir; fosamprenavir;atazanavir; tipranavir; nucleoside and nucleotide reverse transcriptaseinhibitor; zidovudine; didanosine; zalcitabine; stavudine; lamivudine;abacavir; tenofovir disoproxil; adefovir dipivoxil; emtricitabine;entecavir; non-nucleoside reverse transcriptase inhibitor; nevirapine;delavirdine; efavirenz; neuraminidase inhibitor; zanamivir; oseltamivir;moroxydine; inosine pranobex; pleconaril; and enfuvirtide.

Exemplary anti-fungal agents may include but are not limited toallylamine; terbinafine; antimetabolite; flucytosine; azole;fluconazole; itraconazole; ketoconazole; ravuconazole; posaconazole;voriconazole; glucan synthesis inhibitor; caspofungin; micafungin;anidulafungin; polyenes; amphotericin B; amphotericin B ColloidalDispersion (ABCD); and griseofulvin.

Exemplary analgesics may include, but are not limited to opiatederivative, codeine, meperidine, methadone, and morphine.

In various embodiments, docetaxel is the only API in the first liposometype and/or doxorubicin is the only API in the second liposome type.

In various embodiments, the molar ratio of the first lipid layercomponents:doxorubicin is about 100:1 to about 5:1, preferably about20:1 to about 10:1; and the molar ratio of the second lipid layercomponents:docetaxel is about 100:1 to about 5:1, preferably about 20:1to about 10:1.

In various embodiments, the molar ratio of second API (e.g.,doxorubicin):first API (e.g., docetaxel) is about 10:1 to 1:10,preferably about 5:1 to 1:5, and more preferably about 3:1 to 1:3.

The Lipid Layer and Aqueous Solutions

The invention utilizes lipid and aqueous solutions, for example inmaking liposomes in accordance with the invention. Accordingly, thecomposition lipid and/or aqueous solutions can affect the finalcomposition of the liposomes.

In various embodiments, the lipid solution may comprise an organicsolvent. The organic solvent may be a water miscible solvent.Preferably, the water miscible solvent is selected from the groupconsisting of ethanol, methanol, DMSO and isopropanol. Most preferably,the organic solvent is ethanol.

As used herein the term of “cationic lipid” refers to a lipid or acholesterol derivative that carries a net positive charge at about pH3-pH 9.

As used herein the term of “anionic lipid” refers to a lipid or acholesterol derivative that carries a net negative charge at about pH3-pH 9.

As used herein the term “pegylated lipid” refers to a lipid that isconjugated with a polyethylene glycol polymer.

As used herein the term “neutral lipid” refers to the lipid that doesnot carry net charge at about pH 3-pH 9.

The lipid solution may include a mixture of lipids. The mixture oflipids preferably includes cholesterol.

The mixture of lipids may also include a cationic lipid. The cationiclipid may be, but is not limited to, N,N-dioleyl-N,N-dimethylammoniumchloride (“DODAC”); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammoniumchloride (“DOTMA”); N-(2,3-dioleyloxy)propyl)-N,N-dimethylammoniumchloride (“DODMA”); N,N-distearyl-N,N-dimethylammonium bromide (“DDAB”);N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (“DOTAP”);N-(2,3-dioleoyloxy)propyl)-N,N-dimethylammonium chloride (“DODAP”);3-(N—(N′,N′-dimethylaminoethane)carbamoyl)cholesterol (“DC-Chol”);N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (“DMRIE”); 1.2-dilinoleyloxy-N,N-dimethyl-3-aminopropane(DLinDMA); 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA);1.2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA);2-{4-[(3b)-cholest-5-en-3-yloxy]butoxy}-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-amine(CLinDMA).

In some embodiments the mixture of lipids may include an anionic lipid.The anionic lipid may be but is not limited to diacylglycerolphophatidic acid (1,2-distearoyl-sn-glycero-3-phosphate (DSPA);1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA);1,2-dimyristoyl-sn-glycero-3-phosphate (DMPA);1,2-dilauroyl-sn-glycero-3-phosphate (DLPA);1,2-dioleoyl-sn-glycero-3-phosphate (DOPA)), diacylglycerolphosphoglycerol (1,2-distearoyl-sn-glycero-3-phospho-(1′-rac-glycerol)(DSPG); 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DPPG);1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DMPG);1,2-dilauroyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DLPG);1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DOPG)),phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, N-succinylphosphatidylethanolamines, N-glutarylphosphatidylethanolamines,lysylphosphatidylglycerols, and other anionic modifying groups joined toneutral lipids. The mixture of lipids may also include a neutral lipid.The neutral lipid may be but is not limited to diacylglycerolphosphocholine (L-α-phosphatidylcholine, hydrogenated (Soy) (HSPC);diacylglycerol phosphocholine (L-α-phosphatidylcholine, (Soy) (SoyPC)1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC);1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC);1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC);1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC);1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), diacylglycerolphosphoethanolamine (1,2-distearoyl-sn-glycero-3-phosphoethanolamine(DSPE); 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE);1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE);1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE);1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), andphosphatidylserine.

The mixture of lipids may also include a pegylated lipid. The pegylatedlipid may be but is not limited to1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (mPEG-2000-DSPE);1,2-dioctadecanoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (mPEG-2000-DOPE);1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (mPEG-2000-DPPE);1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (mPEG-2000-DMPE);1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (mPEG-2000-DLPE);1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-5000] (mPEG-5000-DSPE);1,2-dioctadecanoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-5000] (mPEG-5000-DOPE);1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-5000] (mPEG-5000-DPPE);1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-5000] (mPEG-5000-DMPE);1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-5000] (mPEG-5000-DLPE).

The mixture of lipid may also include a lipid-like molecule or lipidoid.The mixture of lipid may also include a lipid- or cholesterol-conjugatedmolecule including a protein, or a peptide, or an oligonucleotide.

In various embodiments, the lipid layer includes one or more of thelipid components disclosed herein.

In various embodiments, the first lipid layer and/or the second lipidlayer consist of unsaturated phospholipid and cholesterol.

In various embodiments, the first lipid layer and/or the second lipidlayer consist of unsaturated phospholipid, cholesterol, and pegylatedphospholipid.

In various embodiments, the first lipid layer and the second lipid layercomprise different lipid compositions (e.g., different lipid componentsand/or different amounts of certain lipid components). In variousembodiments, the first liposome and the second liposome are different(e.g., in addition to comprising different APIs, derived from differentlipid solutions and/or aqueous solutions as described below).Alternatively, in some embodiments, the first and second lipid layersare essentially the same (e.g., other than comprising different APIs,comprising essentially the same lipid components and amounts thereof).

In various embodiments, the first lipid layer, the second lipid layer,or both the first and second lipid layers comprise: about 20-75%,preferably about 30-60%, (molar) unsaturated phospholipid; about 10-60%,preferably 20-50%, (molar) cholesterol; and about 0-20%, preferably1-10%, (molar) pegylated phospholipid.

In various embodiments, the molar ratio of the lipid layercomponents:doxorubicin is about 100:1 to about 5:1, preferably about20:1 to about 10:1; and the molar ratio of the lipid layercomponents:docetaxel is about 100:1 to about 5:1, preferably about 20:1to about 10:1.

In various embodiments, the molar ratio of doxorubicin:docetaxel isabout 10:1 to 1:10, preferably about 5:1 to 1:5, and more preferablyabout 3:1 to 1:3.

In various embodiments, the unsaturated phospholipid comprises apolyunsaturated phospholipid or a monounsaturated phospholipid,preferably a phosphatidylcholine, and more preferably and soyphosphatidylcholine or 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine(DOPC).

In various embodiments, the cholesterol comprises a cholesterolderivative, preferably a cationic cholesterol derivative, morepreferably an amino cholesterol derivative, and still more preferablydimethylaminoethanecarbamoyl-cholesterol (DC-cholesterol).

In various embodiments, the pegylated phospholipid comprises aphosphoethanolamine, preferably a1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) and wherein thepegylation is a PEG 500 to PEG 3000, preferably PEG 2000.

In various embodiments, the composition of the lipid layer is tuned toachieve a desired loading of the first drug. Although at least afraction of the first drug is in the lipid layer, one of ordinary skillwill understand that the first drug will have a partition coefficientbetween the lipid layer and aqueous interior. In some embodiments,essentially all of the first drug will be in the lipid layer.

The aqueous solution of the process preferably includes water and abuffer. Buffers may be of but are not limited to phosphate, histidine,HEPES, Tris, acetate, carbonate, and citrate. In various embodiments,the composition of the aqueous solution is tuned to achieve a desiredloading (and/or crystallization) of the second drug. Although at least afraction of the second drug is in the aqueous interior of the liposome,one of ordinary skill will understand that the second drug will have apartition coefficient between the lipid layer and aqueous interior. Insome embodiments, essentially all of the second drug will be in theaqueous interior.

Although, in some embodiments, the first lipid layer and the secondlipid layer have essentially the same lipid compositions, one skilled inthe art will appreciate that, in other embodiments, the first lipidlayer and the second lipid layer can have different lipid compositions(e.g., different lipid components and/or different amounts of certainlipid components).

Methods for Making Liposomes

Examples of apparatuses and methods that can be adapted for making theliposomes of the invention can be found, for example, in U.S. patentapplication Ser. No. 14/209,187 (and published as US20140348900), whichis herein incorporated by reference in its entirety. A description of anumber of different methods of making liposomes in accordance with theinvention are presented in the Examples below.

The invention also provides a method of making the pharmaceuticalcomposition as described or claimed herein. For example, the inventionprovides a method of making a pharmaceutical composition as described orclaimed herein, comprising: (i) making a first liposome type byintroducing a first lipid solution of an unsaturated phospholipid, asterol, docetaxel, and preferably a pegylated phospholipid in ethanolthrough a first port into a mixing chamber and introducing a firstaqueous solution through a second port into the mixing chamber, whereinthe resulting first liposome type does not comprise doxorubicin; (ii)making a second liposome type by introducing a second lipid solution inethanol through a first port into a mixing chamber and introducing asecond aqueous solution through a second port into the mixing chamberand incubating the resulting liposomes with doxorubicin, wherein theresulting second liposome type does not comprise docetaxel; and (iii)combining predetermined amounts of the first liposome type and thesecond liposome type, thereby making the pharmaceutical composition asdescribed or claimed herein.

In various embodiments, the angle between at least one lipid and at oneaqueous solution inlet ports is not 180° or a substantially similarangle. In some aspects, at least one stream of lipid solution and at onestream of aqueous solution collide at an angle less than about 180°.Thus, in some aspects, the method does not include a T-connector.

In some embodiments, the angle between at least one lipid and at oneaqueous solution inlet ports is about 120° or less, e.g., 115° or less,100° or less, 90° or less, 80° or less, 72° or less, 60° or less, 45° orless, 30° or less, 18° or less,

In some embodiments, the aqueous solution in step ii) is introduced viaat least two inlet ports, e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20 or more. In some embodiments, theaqueous solution in step ii) is introduced via at least 3 but no morethan 11 inlet ports, e.g., at least 3 but not more than 7, at least 3but no more than 5, at least 4 but no more than 11, at least 5 but nomore than 11, at least 6 but no more than 11.

In some embodiments, at least two (e.g., 3, 4, 5, 6, 7, etc.) aqueousinlet ports and at least one (e.g., 2, 3, 4, 5, etc.) lipid solutioninlet port are in the same plane.

In some embodiments, at least one (e.g., 2) outlet port is substantiallyperpendicular to the plane of inlet ports. In other embodiments, atleast one (e.g., 2, 3, 4, 5, etc.) outlet port is substantially notperpendicular to the plane of inlet ports.

In some embodiments, at least two (e.g., 3, 4, 5, 6, 7, etc.) aqueoussolution inlet ports and at least one (e.g., 2, 3, 4, 5, etc.) lipidsolution inlet port are not in the same plane.

Preparing Lipid Solutions

The lipid solution may be made from the stock solutions of individuallipids that are mixed together. Lipids are preferably dissolved in anorganic solvent to make a lipid solution. The organic solvent used formaking the lipid solution may be miscible with water. Preferably thesolvent may ethanol, methanol, DMSO, propanol, DMF, THF, acetone,dioxane, ethylene glycol, polyethylene glycol and isopropanol. Morepreferably, the solvent is polyethylene glycol, isopropanol, andethanol. Preferably, the solvent includes less than 10% water. In somecases, the lipid solution may be made from a mixture of lipids,thereupon dissolving the mixture in an organic solvent. Theconcentration of the total lipids in the solution may be in the rangefrom about 1 mg/mL to about 200 mg/mL, e.g., from about 1 mg/mL to about100 mg/mL. More preferably, the concentration of the total lipids in thesolution may be in the range from about 5 mg/mL to about 100 mg/mL orform about 10 mg/mL to 100 mg/mL. In some embodiments, the organicsolvent is ethanol at a concentration of about 70% or more (e.g., 75% ormore, 80% or more, 85% or more, 90% or more, 95% or more, 100%).

The mixture of lipids will be optimized as required for optimal deliveryof the API and is readily optimized by routine experimentation by one ofordinary skill in the art.

In certain embodiments, a water-insoluble API may be dissolved in thelipid solution. The concentration of the API in the lipid solution willdepend on the efficacy of the agent and may easily be determined by oneof ordinary skill in the art. The lipid/API ratio will determined by theencapsulation power of the liposome to the API.

Preparing Aqueous Solutions

A water-soluble API component may be dissolved in a first aqueoussolution (S1). The pH and salinity of the solution may be optimized toaccommodate the requirements for the interaction between the APIcomponent and the lipids to form liposome. These conditions may bereadily determined by one of ordinary skill in the art. Samples areprovided in the Examples below. As will be readily apparent to those ofskill in the art, an aqueous solution that lacks an API, referred to as(S2), may be similar to a solution having the agent. Alternatively, S1and S2 may be different.

Liposome Preparation, Mixing the Solutions

The lipid solution and the aqueous solution(s) preferably enter themanifold from different ports, each with a flow rate of from about 1mL/min to about 6000 mL/min. Preferably, the flow rates may be fromabout 5 mL/min to about 1000 mL/min. More preferably, the rates may befrom about 20 mL/min to about 600 mL/min. In some embodiments, the flowrates are adjusted based on the size of inlet ports to obtain thedesired liposome size, morphology, PDI, and manufacturing scales.

As described above, the first liposome and the second liposome can bedifferent (e.g., different lipid components and/or different amounts ofcertain lipid components, different aqueous interiors, etc.). In suchcases, the first and second liposomes can be made from different lipidsolutions and/or aqueous solutions. However, one skilled in the art willappreciate that in some embodiments, the first and second lipid layerscan be essentially the same.

In some embodiments, the lipid solution and/or the aqueous solution isintroduced via port size of 0.1-5 mm at a flow rate about 1 mL/min toabout 2,500 mL/min.

In some embodiments, the flow velocity of the lipid solution and/or theaqueous solution is from about 0.02 m/s to about 40 m/s, e.g., from 0.1m/s to 30 m/s, from 0.2 m/s to 20 m/s. The flow velocity is adjustedbased on the size of inlet ports to obtain the desired liposome size,morphology, PDI, and manufacturing scale.

Loading of the API into Liposome

In the mixing chamber the lipids are believed to instantaneouslyassemble into liposome particles. When the drug API is carried by thelipid solution or by aqueous solution, it may be encapsulated in theliposome by either lipophilic or electrostatic interaction, or both,between the API and the lipids.

The present invention also provides a method of producing liposome thatdo not contain an API (so-called “empty” liposome). In such embodiments,the API is absent from both the lipid solution and the aqueous solutionthat are mixed in the manifold. The API may be loaded into the liposomesby the process of diffusion or another process. For example, doxorubicinmay be loaded into the liposome with a pH gradient. See U.S. patentapplication Ser. No. 10/019,200, PCT Publication No. WO 2001/005373,U.S. Pat. Nos. 5,785,987, 5,380,531, 5,316,771, and 5,192,549, all ofwhich are incorporated herein by reference.

Preferably, the API is mixed with a liposome solution to upload the APIinto the liposome by diffusion. In one aspect, the API is dissolved inan aqueous solution, and the solution is mixed with the empty liposome.In another aspect, the API may be readily soluble in the solution ofempty liposome, and therefore, the API may be directly mixed with thesolution of the empty liposome.

The volume ratio of the solution of the API to the empty liposomesolution of the API is preferably in the range from about 1:50 to about1:5. A lower volume of the solution is preferred because it avoids asignificant dilution to the final liposome solution.

The drug encapsulation efficiency is preferably greater than 70%. Morepreferably the efficiency is greater than 80%. Most preferably, theefficiency is greater than 90%.

Liposome Concentration Adjustment

Tangent flow filtration may be used to concentrate the liposomesolution.

Buffer Change

Residual organic solvent in the liposome solution may be removed by abuffer change. Preferably, the buffer change is performed by tangentflow filtration. In another embodiment, the buffer change may beperformed by dialysis.

Sterile Filtration

The liposome solutions can be sterilized, for example, by passing thesolution through a 0.22 micron sterile filter.

Liposomes

In various embodiments, the Z-average particle size of the liposomes isfrom about 10 nm to about 2,000 nm, preferably less than 300 nm, morepreferably, the mean particle size may be about 10 to 300 nm or about 20to about 300 nm. Most preferably, the mean particle size is about 20 to120 nm In some embodiments, the liposomes have a polydispersity indexfrom about 0.005 to about 0.8, e.g., 0.005 to about 0.5, 0.01 to about0.5, 0.01 to about 0.4, 0.01 to about 0.2.

Preferably, more than 70% of API is encapsulated in the liposomes. Morepreferably, more than 80% of API is encapsulated in the liposomes, mostpreferably, more than 90% of API is encapsulated in the liposomes.

Optionally, liposomes can be unilamellar. Alternatively, the liposomescan be of multilamellar, or of inverted hexagonal or cubic morphology,or as lipid discs, or hollow liposomes.

Pharmaceutical Compositions

In various embodiments, the pharmaceutical composition is for use as amedicament. In various embodiments, the pharmaceutical composition isfor use as a cancer therapeutic. In various embodiments, thepharmaceutical composition comprises an antibiotic, antiviral,anti-diabetes, anti-hypertension, anti-fungal, or analgesic drug.

The invention also provides a pharmaceutical composition consisting of:(i) a plurality of liposomes of the first type as described or claimedherein; (ii) a plurality of liposomes of the second type as described orclaimed herein; and (iii) one or more pharmaceutical excipients.

In various embodiments, the plurality of liposomes are comprised in aninjectable formulation, for example, by subcutaneous, intravenous,intramuscular, intrathecal or intraperitoneal injection. Injectableformulations can be aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution, Ringer's solution, orphysiological saline buffer. The injectable formulation can containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the liposomes can be in a dried or powder formfor constitution with a suitable vehicle, e.g., sterile pyrogen-freewater, before use.

Treatment and Administration

The invention provides a method of treating a subject comprisingadministering an effective amount of any of the pharmaceuticalcompositions of the invention described or claimed herein. In variousembodiments, the subject has a cancer. In various embodiments, thecancer is a lung cancer, preferably non-small cell lung cancer (NSCLC);colon cancer; breast cancer; or liver cancer, preferably hepatocellularcarcinoma (HCC).

Accordingly, the invention provides methods for treating cancer cellsand/or tissue, including cancer cells and/or tissue in a human subject.Cancer can be caused by malignant tumors formed by an abnormal growth ofcells and tissue leading to organ failure.

Solid tumors can be neoplasms (new growth of cells) or lesions (damageof anatomic structures or disturbance of physiological functions) formedby an abnormal growth of body tissue cells other than blood, bone marrowor lymphatic cells. A solid tumor consists of an abnormal mass of cellswhich may stem from different tissue types such as liver, colon, breast,or lung, and which initially grows in the organ of its cellular origin.However, such cancers may spread to other organs through metastatictumor growth in advanced stages of the disease.

The subject being treated may have been diagnosed with cancer. Thesubject may have locally advanced, unresectable, or metastatic cancerand/or may have failed a prior first-line therapy. In variousembodiments, the cancer is liver cancer (e.g., hepatocellular carcinoma,HCC). In various embodiments, the liver cancer (e.g., HCC) can beintermediate, advanced, or terminal stage. The liver cancer (e.g., HCC)can be metastatic or non-metastatic. Liver cancer can include a livertumor resulting from the metastasis of a non-liver cancer, to the liver.The liver cancer (e.g., HCC) can be resectable or unresectable. Theliver cancer (e.g., HCC) can comprise a single tumor, multiple tumors,or a poorly defined tumor with an infiltrative growth pattern (intoportal veins or hepatic veins). The liver cancer (e.g., HCC) cancomprise a fibrolamellar, pseudoglandular (adenoid), pleomorphic (giantcell), or clear cell pattern. The liver cancer (e.g., HCC) can comprisea well differentiated form, and tumor cells resemble hepatocytes, formtrabeculae, cords, and nests, and/or contain bile pigment in cytoplasm.The liver cancer (e.g., HCC) can comprise a poorly differentiated form,and malignant epithelial cells are discohesive, pleomorphic, anaplastic,and/or giant. In some embodiments, the liver cancer (e.g., HCC) isassociated with hepatitis B, hepatitis C, cirrhosis, or type 2 diabetes.

In various embodiments, the cancer is a lung cancer, preferablynon-small cell lung cancer (NSCLC); colon cancer; breast cancer; orliver cancer, preferably hepatocellular carcinoma (HCC).

In various embodiments, the docetaxel can be in a concentration of 10,20, 30, 40, 50, 75, 80, 100, 125, 150, or 160 mg/mL. A dose can be about10 mg/m² to 150 mg/m² (e.g., 10, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90,100, 110, 120, 125, 130, 140, or 150 mg/m²). For example, a dose can be75 mg/m². A dose can be administered every 3 weeks for 1, 2, 3, 5, 5, or6 cycles. One skilled in the art will appreciate that dosing guidelinesfor docetaxel are known in the art, and can be adapted based uponfactors including, but not limited to the cancer type, the cancer stage,the dosing regimen, the dose of doxorubicin, and/or the efficacy of thepharmaceutical compositions of the invention.

In various embodiments, the doxorubicin can be in a concentration of0.1, 0.5, 1, 1.5, 2, 3, 4, or 5 mg/mL. A dose can be about 1 mg/m² to100 mg/m² (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30, 40, 50, 60,70, 75, 80, 90, or 100 mg/m²). For example, a dose can be 30 mg/m². Adose can be administered every 3 weeks for 1, 2, 3, 5, 5, or 6 cycles.One skilled in the art will appreciate that dosing guidelines fordocetaxel are known in the art, and can be adapted based upon factorsincluding, but not limited to the cancer type, the cancer stage, thedosing regimen, the dose of doxorubicin, and/or the efficacy of thepharmaceutical compositions of the invention.

In various embodiments, the liposome of the first API (e.g. docetaxel)and the liposome of the second API (e.g. doxorubicin) are mixed andco-administered to a subject.

In various embodiments, the liposome of the first API (e.g. docetaxel)and the liposome of the second API (e.g. doxorubicin) are administeredseparately in sequence.

The following examples are illustrative and not restrictive. Manyvariations of the technology will become apparent to those of skill inthe art upon review of this disclosure. The scope of the technologyshould, therefore, be determined not with reference to the examples, butinstead should be determined with reference to the appended claims alongwith their full scope of equivalents.

EXAMPLES Example 1 Preparation of Liposomal formulation CPT307A

CPT307A comprises a nonsaturated lipid1,2-Dioleoyl-sn-glycero-3-Phosphatidylcholine (DOPC), cholesterol, and1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy9polyethyleneglycol)-2000].A lipid solution in ethanol was made by dissolving 1200 mg of1,2-Dioleoyl-sn-glycero-3-Phosphatidylcholine (DOPC), 160 mg ofcholesterol, and 400 mg of1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy9polyethyleneglycol)-2000](mPEG2000-DSPE) in 40 mL of anhydrous ethanol. In addition, threeaqueous solutions of 250 mM ammonium sulfate, pH 6.5 were used. Twentymilliliter of each of the above four solutions was loaded into a 20 mLsyringe. Each syringe was connected to an inlet port of a five-portmanifold by tubing. Through the tubing, the solutions in the syringeswere pumped into the mixing chamber of the manifold by a syringe pump.The liposome solution exited through an outlet port and was collected ina glass bottle and was then concentrated by tangent flow filtration. Thebuffer was changed into a histidine/sucrose buffer (10 mM histidine,9.2% sucrose, pH 6.5) by tangent flow filtration.

Liposomal formulation CPT307A was prepared by loading doxorubicin (DXR)into the empty liposomes. Twenty eight milliliters of the emptyliposomes was mixed with 6.85 mL of a DXR solution in thehistidine/sucrose buffer at a concentration of 8.93 mg/mL, and incubatedat 42° C. for 3 hours, 99.8% of DXR was encapsulated. The encapsulatedliposome was then sterilized by filtration through a 0.22 μm filter. Thecomposition (% molar) of the CPT307A lipid solution is illustrated inTable 1. The final Z-average particle size of the loaded liposome was37.9 nm, with a DXR concentration of 1.74 mg/mL. The volume-weightedparticle size distribution of CPT307A determined by dynamic lightscattering (Malvern Zetasizer Nano ZS) is shown in FIG. 1.

TABLE 1 Lipid Composition of Example 1. Component CPT307A % (molar) *DOPC 73.6 Cholesterol 20 mPEG2000-DSPE 6.4 DXR 13.1 * The valuerepresents the molar % of each component vs. total lipids.

Example 2 Preparation of Liposomal Formulation CPT307B

The lipid composition of CPT307B is identical to CPT307A, it comprisesDOPC, cholesterol, and mPEG2000-DSPE. It was found that compared to asaturated lipid such as DSPC, DPPC, HSPC, the nonsaturated lipid DOPCmakes liposome a greater capacity to encapsulate docetaxel. To makeCPT307B, a lipid/DOCE solution was prepared by dissolving 2100 mg ofDOPC, 280 mg of cholesterol, 700 mg of mPEG2000-DSPE, and 175 mg ofdocetaxel (DOCE) in 70 mL of anhydrous ethanol. The composition (%molar) of the CPT307B lipid solution is illustrated in Table 2. Inaddition, three aqueous solutions of 250 mM ammonium sulfate, pH 6.5were used. Twenty milliliter of each of the above four solutions wasloaded into a 20 mL syringe. Each syringe was connected to an inlet portof a five-port manifold by tubing. Through the tubing, the solutions inthe syringes were pumped into the mixing chamber of the manifold by asyringe pump. The liposome solution exited through an outlet port andwas collected in a glass bottle and was then concentrated by tangentflow filtration. The buffer was changed into a histidine/sucrose buffer(10 mM histidine, 9.2% sucrose, pH 6.5) by tangent flow filtration. Theformulation was then sterilized by filtration through a 0.22 μm filter.The Z-average particle size was 32.9 nm for CPT307B. The volume-weightedparticle size distribution of CPT307B determined by dynamic lightscattering (Malvern Zetasizer Nano ZS) is shown in FIG. 2.

TABLE 2 Lipid Composition of Example 2. Component CPT307B % (molar)*DOPC 73.6 Cholesterol 20 mPEG2000-DSPE 6.4 DOCE 6.0 *The valuerepresents the molar % of each component vs. total lipids.

Example 3 Preparation of Combination Liposomal Formulation CPT307AB

Liposomal formulation CPT307AB was prepared by mixing CPT307A (seeExample 1) and CPT307B (see Example 2) at a 1:1 molar ratio (or 1:1.5w/w ratio) of DXR encapsulated in CPT307A and DOCE encapsulated inCPT307B. For instance, CPT307AB was prepared by mixing 12.07 mL ofCPT307A containing 1.74 mg/mL of DXR and 12.26 mL of CPT307B containing2.57 mg/mL of DOCE. The mixture was gently swirled to allow for completemixing. The composition (% molar) of the CPT307AB lipid solution isillustrated in Table 3. The final concentrations of DXR and DOCE inCPT307AB were 0.86 mg/mL and 1.29 mg/mL, respectively.

TABLE 3 Lipid Composition of Example 3. Component CPT307AB % (molar)*DOPC 73.6 Cholesterol 20 mPEG2000-DSPE 6.4 DXR 4.1 DOCE 4.1 *The valuerepresents the molar % of each component vs. total lipids.

Example 4 Preparation of Liposomal Formulation CPT319A

CPT319A comprises of DOPC, cholesterol,3β-[N—(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride(DC-Cholesterol), and mPEG2000-DSPE. DC-Cholesterol is a cationicderivative of cholesterol that brings positive surface charges toliposome and thus dramatically the in vivo distribution of the liposome.To make the liposome, a lipid solution was prepared firstly bydissolving 1800 mg of DOPC, 300 mg of cholesterol, 420 mg ofDC-cholesterol and 600 mg of mPEG2000-DSPE in 60 mL of anhydrousethanol. In addition, three aqueous solutions of 250 mM ammoniumsulfate, pH 6.5 were used. Twenty milliliter of each of the above foursolutions was loaded into a 20 mL syringe. Each syringe was connected toan inlet port of a five-port manifold by tubing. Through the tubing, thesolutions in the syringes were pumped into the mixing chamber of themanifold by a syringe pump. The liposome solution exited through anoutlet port was collected in a glass bottle and was then concentrated bytangent flow filtration. The buffer was changed into a histidine/sucrosebuffer (10 mM histidine, 9.2% sucrose, pH 6.5) by tangent flowfiltration.

Liposomal formulation CPT319A was prepared by loading DXR into the emptyliposomes. Thirty milliliters of the empty liposomes containing lipidsat 33.5 mg/mL was mixed with 83.6 mg of DXR that had been pre-dissolvedin the histidine/sucrose buffer, and incubated at 42° C. for 3 hours,94.6% of DXR was encapsulated. The encapsulated liposome was thensterilized by filtration through a 0.22 μm filter. The composition (%molar) of the CPT319A illustrated in Table 4. The final Z-average of theparticle size of the loaded liposome was 43.6 nm. The volume-weightedparticle size distribution of CPT319A determined by dynamic lightscattering (Malvern Zetasizer Nano ZS) is shown in FIG. 3.

TABLE 4 Lipid Composition of Example 4. Component CPT319A % (molar)*DOPC 61.7 Cholesterol 17.7 DC-Cholesterol 15.2 mPEG2000-DSPE 5.4 DXR10.3 *The value represents the molar % of each component vs. totallipids.

Example 5 Preparation of Liposomal Formulation CPT319B

CPT319B comprises of DOPC, cholesterol,3β-[N—(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride(DC-Cholesterol), and mPEG2000-DSPE. DC-Cholesterol is a cationicderivative of cholesterol that brings positive surface charges toliposome and thus dramatically the in vivo distribution of the liposome.The lipids composition is identical to CPT319A. To make CPT319B, alipid/DOCE solution was prepared by dissolving 1848 mg of DOPC, 303 mgof cholesterol, 423 mg of DC-Cholesterol, 605 mg of mPEG2000-DSPE, and154 mg of DOCE in 61.5 mL of anhydrous ethanol. The composition (%molar) of the CPT319B lipid solution is illustrated in Table 5. Inaddition, three aqueous solutions of 250 mM ammonium sulfate, pH 6.5were used. Twenty milliliter of each of the above four solutions wasloaded into a 20 mL syringe. Each syringe was connected to an inlet portof a five-port manifold by tubing. Through the tubing, the solutions inthe syringes were pumped into the mixing chamber of the manifold by asyringe pump. The liposome solution exited through an outlet port wascollected in a glass bottle and was then concentrated by tangent flowfiltration. The buffer was changed into a histidine/sucrose buffer (10mM histidine, 9.2% sucrose, pH 6.5) by tangent flow filtration. Theformulation was then sterilized by filtration through a 0.22 μm filter.The volume-weighted particle size distribution of CPT319B determined bydynamic light scattering testing with a Malvern Zetasizer Nano ZS isshown in FIG. 4. The Cryo-TEM images of CPT319B are shown in FIG. 5. Theparticles exhibited overwhelmingly spherical unilamellar morphology withhomogenous particle size. The negative stained TEM image of CPT319B isshown in FIG. 6, which further presents the small spherical andhomogenous particles.

TABLE 5 Lipid Composition of Example 5. Component CPT319B % (molar)*DOPC 61.7 Cholesterol 17.7 DC-Cholesterol 15.2 mPEG2000-DSPE 5.4 DOCE3.5 *The value represents the molar % of each component vs. totallipids.

Example 6 Preparation of Combination Liposomal Formulation CPT319AB

Liposomal formulation CPT319AB was prepared by mixing CPT319A (seeExample 4) and CPT319B (see Example 5) at a 1:1 molar ratio (or 1:1.5w/w ratio) of DXR encapsulated in CPT319A and DOCE encapsulated inCPT319B. For instance, CPT319AB was prepared by mixing 13.7 mL ofCPT319A containing 1.24 mg/mL of DXR and 11.7 mL of CPT319B containing2.18 mg/mL of DOCE. The mixture was gently swirled to allow for completemixing. The composition (% molar) of the CPT319AB lipid solution isillustrated in Table 6. The final concentrations of DXR and DOCE inCPT319AB were 0.67 mg/mL and 1.0 mg/mL, respectively.

TABLE 6 Lipid Composition of Example 6. Component CPT319AB % (molar)*DOPC 61.7 Cholesterol 17.7 DC-Cholesterol 15.2 mPEG2000-DSPE 5.4 DXR 2.6DOCE 2.6 *The value represents the molar % of each component vs. totallipids.

Example 7 Preparation of Liposomal Formulation CPT317A

CPT317A comprises of the same type of lipids as CPT319A while lipidsratio is changed (increased cholesterol and decreased DOPC andDC-cholesterol). To make CPT317A, a lipid solution was prepared bydissolving 600 mg of DOPC, 140 mg of cholesterol, 84 mg ofDC-Cholesterol, and 200 mg of mPEG2000-DSPE in 20 mL of anhydrousethanol. In addition, three aqueous solutions of 250 mM ammoniumsulfate, pH 6.5 were used. Twenty milliliter of each of the above foursolutions was loaded into a 20 mL syringe. Each syringe was connected toan inlet port of a five-port manifold by tubing. Through the tubing, thesolutions in the syringes were pumped into the mixing chamber of themanifold by a syringe pump. The liposome solution exited through anoutlet port was collected in a glass bottle and was then concentrated bytangent flow filtration. The buffer was changed into a histidine/sucrosebuffer (10 mM histidine, 9.2% sucrose, pH 6.5) by dialysis.

Liposomal formulation CPT317A was prepared by loading DXR into the emptyliposomes. Fifteen milliliters of the empty liposomes containing lipidsat 40.6 mg/mL was mixed with 50.7 mg of DXR and incubated at 42° C. for3 hours, 99.2% of DXR was encapsulated. The encapsulated liposome wasthen sterilized by filtration through a 0.22 μm filter. The Z-averageparticle size of the loaded liposome determined by dynamic lightscattering (Malvern Zetasizer Nano ZS) was 37.6 nm. The composition (%molar) of the CPT317A is illustrated in Table 7,

TABLE 7 Lipid Composition of Example 7. Component CPT317A % (molar)*DOPC 56.6 Cholesterol 26.9 DC-Cholesterol 11.6 mPEG2000-DSPE 5.0 DXR11.6 *The value represents the molar % of each component vs. totallipids.

Example 8 Preparation of Liposomal Formulation CPT317B

The liposome composition of CPT317B is identical to CPT317A. To makeCPT317B, a lipid/DOCE solution was prepared by dissolving 600 mg ofDOPC, 140 mg of cholesterol, 84 mg of DC-Cholesterol, 200 mg ofmPEG2000-DSPE, and 50 mg of DOCE in 20 mL of anhydrous ethanol. Thecomposition (% molar) of the CPT317B is illustrated in Table 8. Inaddition, three aqueous solutions of 250 mM ammonium sulfate, pH 6.5were used. Twenty milliliter of each of the above four solutions wasloaded into a 20 mL syringe. Each syringe was connected to an inlet portof a five-port manifold by tubing. Through the tubing, the solutions inthe syringes were pumped into the mixing chamber of the manifold by asyringe pump. The liposome solution exited through an outlet port wascollected in a glass bottle and was then concentrated by tangent flowfiltration. The buffer was changed into a histidine/sucrose buffer (10mM histidine, 9.2% sucrose, pH 6.5) by tangent flow filtration. Theformulation was then sterilized by filtration through a 0.22 μm filter.The Z-average particle size determined by dynamic light scattering(Malvern Zetasizer Nano ZS) was 47.9 nm.

TABLE 8 Lipid Composition of Example 8. Component CPT317B % (molar)*DOPC 56.6 Cholesterol 26.9 DC-Cholesterol 11.6 mPEG2000-DSPE 5.0 DOCE4.6 *The value represents the molar % of each component vs. totallipids.

Example 9 Preparation of Combination Liposomal Formulation CPT317AB

Liposomal formulation CPT317AB was prepared by mixing CPT317A (seeExample 7) and CPT317B (see Example 8) at a 1:1 molar ratio (or 1:1.5w/w ratio) of DXR encapsulated in CPT317A and DOCE encapsulated inCPT317B. For instance, CPT317AB was prepared by mixing 2.48 mL ofCPT317A containing 1.43 mg/mL of DXR and 2.86 mL of CPT317B containing1.86 mg/mL of DOCE. The mixture was gently swirled to allow for completemixing. The composition (% molar) of the CPT317AB lipid solution isillustrated in Table 9. The final concentrations of DXR and DOCE inCPT317AB were 0.66 mg/mL and 1.0 mg/mL, respectively.

TABLE 9 Lipid Composition of Example 9. Component CPT317AB % (molar)DOPC 56.6 Cholesterol 26.9 DC-Cholesterol 11.6 mPEG2000-DSPE 5.0 DXR 3.3DOCE 3.3 *The value represents the molar % of each component vs. totallipids.

Example 10 Preparation of Liposomal Formulation CPT308B

Different from other exemplary formulations above those contain amonounsaturated lipid DOPC, CPT308B contains a polyunsaturated lipid-SoyPC. It was found that compared to DOPC, Soy PC further increases DOCEencapsulation capacity of liposome. To make CPT308B, two milliliters ofthe lipids/DOCE solution was prepared by first dissolving 30 mg ofL-α-phosphatidylcholine (Soy PC), 10 mg of cholesterol, 10 mg ofmPEG2000-DSPE, and 6 mg of DOCE in 2 mL of anhydrous ethanol. Thecomposition (% molar) of the lipid formulation of CPT308B is illustratedin Table 10. In addition, three aqueous solutions of 250 mM ammoniumsulfate, pH 6.5 were used. Two milliliters of each of the above foursolutions was loaded into a 20 mL syringe. Each syringe was connected toan inlet port of a five-port manifold by tubing. Through the tubing, thesolutions in the syringes were pumped into the mixing chamber of themanifold by a syringe pump. The liposome solution exited through anoutlet port was collected in a glass vial. The buffer was changed into ahistidine/sucrose buffer (10 mM histidine, 9.2% sucrose, pH 6.5) bydialysis. The formulation was then sterilized by filtration through a0.22 μm filter. The Z-average particle size of CPT308B determined bydynamic light scattering (Malvern Zetasizer Nano ZS) was 34.7 nm.

TABLE 10 Lipid Composition of Example 10. Component CPT308B % (molar)*Soy PC 57.0 Cholesterol 38.1 mPEG2000-DSPE 4.9 DOCE 10.9 *The valuerepresents the molar % of each component vs. total lipids.

Example 11 Preparation of Liposomal Formulation CPT309B

In order to further improve DOCE encapsulation capacity of the liposomecontaining Soy PC, the molar ratio of Soy PC in CPT309B is increasedwhile the molar ratio of cholesterol is decreased. To make CPT309B, twomilliliters of the lipids/DOCE solution was prepared by dissolving 30 mgof Soy PC, 4 mg of cholesterol, 10 mg of mPEG2000-DSPE, and 6 mg of DOCEin 2 mL anhydrous ethanol. The composition (% molar) of the lipidformulation CPT309B lipid solution is illustrated in Table 11. Inaddition, three aqueous solutions of 250 mM ammonium sulfate, pH 6.5were used. Two milliliters of each of the above four solutions wasloaded into a 20 mL syringe. Each syringe was connected to an inlet portof a five-port manifold by tubing. Through the tubing, the solutions inthe syringes were pumped into the mixing chamber of the manifold by asyringe pump. The liposome solution exited through an outlet port wascollected in a glass vial. The buffer was changed into ahistidine/sucrose buffer (10 mM histidine, 9.2% sucrose, pH 6.5) bydialysis. The formulation was then sterilized by filtration through a0.22 μm filter. The Z-average particle size of CPT309B determined bydynamic light scattering (Malvern Zetasizer Nano ZS) was 35.0 nm.

TABLE 11 Lipid Composition of Example 11. Component CPT309B % (molar)Soy PC 73.9 Cholesterol 19.7 mPEG2000-DSPE 6.4 DOCE 14.2 *The valuerepresents the molar % of each component vs. total lipids.

Example 12 Preparation of Liposomal Formulation CPT313B

In CPT313B, cholesterol is completely substituted by DC-cholesterol. Tomake CPT313B, a lipid/DOCE solution was prepared by dissolving 33 mg ofSoy PC, 20.5 mg of DC-Cholesterol, 11 mg of mPEG2000-DSPE, and 4.4 mg ofDOCE in 2.2 mL of anhydrous ethanol. The composition (% molar) of theCPT313B is illustrated in Table 12. In addition, three aqueous solutionsof 250 mM ammonium sulfate, pH 6.5 were used. Two milliliters of each ofthe above four solutions was loaded into a 20 mL syringe. Each syringewas connected to an inlet port of a five-port manifold by tubing.Through the tubing, the solutions in the syringes were pumped into themixing chamber of the manifold by a syringe pump. The liposome solutionexited through an outlet port was collected in a glass vial. The bufferwas changed into a histidine/sucrose buffer (10 mM histidine, 9.2%sucrose, pH 6.5) by dialysis. The formulation was then sterilized byfiltration through a 0.22 μm filter. The Z-average particle size ofCPT313B determined by dynamic light scattering (Malvern Zetasizer NanoZS) was 37.4 nm.

TABLE 12 Lipid Composition of Example 12. Component CPT313B % (molar)*Soy PC 50.4 mPEG2000-DSPE 4.4 DC-cholesterol 45.2 DOCE 6.5 *The valuerepresents the molar % of each component vs. total lipids.

Example 13 Preparation of Liposomal Formulation CPT323B

CPT323C comprises of DOPC, cholesterol, DC-cholesterol but in theabsence of pegylated lipid, indicating that pegylated lipid is optionalto the formulation. To make CPT323B, a lipid/DOCE solution was preparedby dissolving 300 mg of DOPC, 50 mg of cholesterol, 70 mg ofDC-Cholesterol, and 25 mg of DOCE in 10 mL of anhydrous ethanol. Thecomposition (% molar) of the CPT323B is illustrated in Table 13. Inaddition, three aqueous solutions of 250 mM ammonium sulfate, pH 6.5were used. Ten milliliter of each of the above four solutions was loadedinto a 20 mL syringe. Each syringe was connected to an inlet port of afive-port manifold by tubing. Through the tubing, the solutions in thesyringes were pumped into the mixing chamber of the manifold by asyringe pump. The liposome solution exited through an outlet port wascollected in a glass bottle and was then concentrated by tangent flowfiltration. The buffer was changed into a histidine/sucrose buffer (10mM histidine, 9.2% sucrose, pH 6.5) by dialysis. The formulation wasthen sterilized by filtration through a 0.22 μm filter. The Z-averageparticle size determined by dynamic light scattering (Malvern ZetasizerNano ZS) of CPT323B was 47.5 nm.

TABLE 13 Lipid Composition of Example 13. Component CPT323B % (molar)*DOPC 59.5 Cholesterol 20.1 DC-Cholesterol 20.3 DOCE 4.8 *The valuerepresents the molar % of each component vs. total lipids.

Example 14 Preparation of Liposomal Formulation CPT324B

Different from other exemplary formulations, CPT324B contains apolyunsaturated lipid-Soy PC for a higher DOCE capacity, and a cationiclipid DOTAP other than DC-cholesterol. To make CPT324B, a lipid/DOCEsolution was prepared by dissolving 60 mg of Soy PC, 40 mg ofcholesterol, 60 mg of 1,2-dioleoyl-3-trimethylammonium-propane (chloridesalt) (DOTAP), 40 mg of mPEG2000-DSPE, and 25 mg of DOCE in 10 mL ofanhydrous ethanol. The composition (% molar) of the CPT324B isillustrated in Table 14. In addition, three aqueous solutions of 250 mMammonium sulfate, pH 6.5 were used. Ten milliliter of each of the abovefour solutions was loaded into a 20 mL syringe. Each syringe wasconnected to an inlet port of a five-port manifold by tubing. Throughthe tubing, the solutions in the syringes were pumped into the mixingchamber of the manifold by a syringe pump. The liposome solution exitedthrough an outlet port was collected in a glass bottle and was thenconcentrated by tangent flow filtration. The buffer was changed into ahistidine/sucrose buffer (10 mM histidine, 9.2% sucrose, pH 6.5) bydialysis. The formulation was then sterilized by filtration through a0.22 μm filter. The Z-average particle size of CPT324B determined bydynamic light scattering (Malvern Zetasizer Nano ZS) was 39.0 nm.

TABLE 14 Lipid Composition of Example 14. Component CPT324B % (molar)*Soy PC 27.6 Cholesterol 36.9 mPEG2000-DSPE 4.8 DOTAP 30.7 DOCE 11.0 *Thevalue represents the molar % of each component vs. total lipids.

Example 15 Combination Liposome CPT319AB Augments Efficacy AgainstNon-Small Cell Lung Cancer (NSCLC)

Female Balb/c nude mice ranging from 6-8 weeks of age were inoculatedsubcutaneously on the right flank with NSCLC cell line A549 tumor cells(1×10⁷ cells/mouse) in 0.1 mL phosphate buffered saline (PBS) for tumordevelopment. On Day 16 following tumor cell inoculation (tumor size wasapproximately 117 mm³), treatments were started with formulations ofCPT319A at 5 mg/kg doxorubicin, CPT319B at 7.5 mg/kg docetaxel, CPT319ABat 5 mg/kg doxorubicin/7.5 mg/kg docetaxel, or the non-liposomalcombination formulation of 5 mg/kg doxorubicin/7.5 mg/kg docetaxel byintravenous (IV) injection through the tail vein. Three additionaltreatments were administered on Day 20, Day 27, and Day 34. The studywas terminated on Day 45. The tumor growth curves and tumor weightinhibition percentages (TW Inh %) on Day 45 of the formulations comparedto the PBS control group are shown in FIG. 7. All of the liposomalformulations were more efficacious than the non-liposomal combination ofDOCE/DXR. In addition, the combination liposome, CPT319AB was the mostefficacious in this example. Compared to the PBS control group, thecombination liposomal formulation CPT319AB reduced 81% of the tumorweight that was significantly more efficacious than the 51% of DOCEliposome CPT319B, 27% of DXR liposome CPT319A, and 17% of thenon-liposomal combination of DOCE/DXR.

Example 16 Combination Liposome CPT307AB Augments Efficacy Against NSCLC

Female Balb/c nude mice ranging from 6-8 weeks of age were inoculatedsubcutaneously on the right flank with NSCLC cell line A549 tumor cells(1×10⁷ cells/mouse) in 0.1 mL phosphate buffered saline (PBS) buffer fortumor development. On Day 16 following tumor cell inoculation (tumorsize was approximately 117 mm³), treatments were started withformulations of CPT307A at 5 mg/kg doxorubicin, CPT307B at 7.5 mg/kgdocetaxel, CPT307AB at 5 mg/kg doxorubicin/7.5 mg/kg docetaxel, or thenon-liposomal combination formulation of 5 mg/kg doxorubicin/7.5 mg/kgdocetaxel by intravenous (IV) injection through the tail vein. Threeadditional treatments were administered on Day 20, Day 27, and Day 34.The study was terminated on Day 45. The tumor growth curves and tumorweight inhibition percentages (TW Inh %) on Day 45 of the formulationscompared to the PBS control group are shown in FIG. 8. All of theliposomal formulations were more efficacious than the non-liposomalcombination of DOCE/DXR. In addition, the combination liposome, CPT307ABwas the most efficacious in this example. Compared to the PBS controlgroup, the combination liposomal formulation CPT307AB reduced 72% of thetumor weight that was significant more efficacious than the 44% of DOCEliposome CPT307B, 11% of DXR liposome CPT307A, and 17% of thenon-liposomal combination of DOCE/DXR.

Example 17 Antitumor Activity of Combination Liposome CPT319AB AgainstHuman Colon Cancer in Xenograft Mouse Model

Female Balb/c nude mice ranging from 6-8 weeks of age were inoculatedsubcutaneously at the right flank with human colon cancer cell lineHCT-116 tumor cells (5×10⁶ cells/mouse) in 0.1 mL PBS buffer for tumordevelopment. On Day 9 following tumor cell inoculation (tumor size wasapproximately 141 mm³), treatments were started with formulations ofCPT319AB at 3 different doses: 5 mg/kg doxorubicin/7.5 mg/kg docetaxel,2.5 mg/kg doxorubicin/3.75 mg/kg docetaxel, or 1.25 mg/kgdoxorubicin/1.875 mg/kg docetaxel by intravenous (IV) injection throughthe tail vein. Two additional treatments were administered on Day 16 andDay 23. The study was terminated on Day 37. The tumor growth curvesshown in FIG. 9 illustrate dose responses of the HCT-116 tumor cells tothe liposomal formulations (which are compared to the vehicle control).Compared to the vehicle control, tumor weight was inhibited by 59, 52,and 29% in the treatment groups (doses from high to low).

Example 18 Antitumor Activity of Combination Liposome CPT307AB AgainstHuman Colon Cancer in Xenograft Mouse Model

Female Balb/c nude mice ranging from 6-8 weeks of age were inoculatedsubcutaneously at the right flank with human colon cancer cell lineHCT-116 tumor cells (5×10⁶ cells/mouse) in 0.1 mL PBS buffer for tumordevelopment. On Day 9 following tumor cell inoculation (tumor size wasapproximately 141 mm³), treatments were started with formulations ofCPT307AB at 3 different doses: 5 mg/kg doxorubicin/7.5 mg/kg docetaxel,2.5 mg/kg doxorubicin/3.75 mg/kg docetaxel, or 1.25 mg/kgdoxorubicin/1.875 mg/kg docetaxel by intravenous (IV) injection throughthe tail vein. Two additional treatments were administered on Day 16 andDay 23. The study was terminated on Day 37. The tumor growth curvesshown in FIG. 10 illustrate dose responses of the HCT-116 tumor cells tothe liposomal formulations (which are compared to the vehicle control).Compared to the vehicle control, tumor weight was inhibited by 64, 52,and 45% in the treatment groups (doses from high to low).

Example 19 Antitumor Activity of Combination Liposome CPT319AB AgainstHuman Primary Hepatocellular Carcinoma in Xenograft Mouse Model

Female SCID Beige mice ranging from 6-8 weeks of age were inoculatedsubcutaneously at the right flank with fragments of human primaryhepatocellular carcinoma tumor (P3 WP HCC) for tumor development. On Day32 after tumor inoculation (tumor size was approximately 143 mm³),treatments were started with a formulation of CPT319AB at 5 mg/kgdoxorubicin/7.5 mg/kg docetaxel by intravenous (IV) injection throughthe tail vein. Two additional treatments were made on Day 39 and Day 46.The study was terminated on Day 63. The tumor growth curves are shown inFIG. 11. CPT319AB reduced tumor weight by 68% compared to the vehiclecontrol.

Example 20 Cationic Lipid DC-Cholesterol Enhances the Antitumor Activityof Liposomes Against NSCLC

Female Balb/c nude mice ranging from 6-8 weeks of age were inoculatedsubcutaneously at the right flank with NSCLC cell line A549 tumor cells(1×10⁷ cells/mouse) in 0.1 mL PBS buffer for tumor development. On Day16 following tumor cell inoculation (tumor size was approximately 117mm³), treatments were started with formulations of CPT307A or CPT319A at5 mg/kg doxorubicin, CPT307B or CPT319B at 7.5 mg/kg docetaxel, CPT307ABor CPT319AB at 5 mg/kg doxorubicin/7.5 mg/kg docetaxel, or thenon-liposomal combination formulation of 5 mg/kg doxorubicin/7.5 mg/kgdocetaxel by intravenous (IV) injection through the tail vein. Threeadditional treatments were made on Day 20, Day 27, and Day 34. The studywas terminated on Day 45. The lipid compositions of the CPT307 (withoutDC-Cholesterol) and CPT319 (with DC-Cholesterol) formulations are shownin Table 15. The tumor growth curves are shown in FIG. 12. The tumorweight inhibition % on Day 45 is shown in Table 16.

The tumor inhibition rank order (from high to low) was:CPT319AB>CPT307AB>CPT319B>CPT307B>CPT319A>CPT307A>PBS. For eachinstance, CPT319 (with DC-Cholesterol) was more efficacious than CPT307(without DC-Cholesterol). This indicates that the cationic lipidDC-cholesterol in CPT319 enhances the antitumor activity of the liposomeformulations.

TABLE 15 Lipid Compositions of Example 20. DC-cholesterol CholesterolmPEG-DSPE DOPC Formulation (molar %) (molar %) (molar %) (molar %)CPT319 15 18 5 62 CPT307 0 24 6 70

TABLE 16 Tumor Weight Inhibition % on Day 45 of Example 20 Formulation AB AB CPT319 27% 51% 81% CPT307 11% 44% 72%

Example 21 Cationic Lipid DC-Cholesterol Increases the Half-Life(t_(1/2)) of DXR

Male CD-1 mice ranging from 20-25 g body weight were split up intogroups of three. Each mouse was administered with a single dose ofCPT319A or CPT307A at 5 mg/kg DXR by intravenous (IV) injection throughthe tail vein. A non-liposomal combination of DXR was used as thecontrol. Blood samples were collected at 0.167, 1, 3, 8, 24, and 48 hafter the injection. DXR plasma concentration was determined by liquidchromatography-tandem mass spectrometry (LC-MS-MS). The plasmaconcentration curves of DXR are shown in FIG. 13 and the t_(1/2) andarea under the plasma concentration time curve (AUC) are provided inTable 17. *BLOQ: Below Limit of Quantitation. The non-liposomal DXR wascleared quickly from the blood and resulted in a very low AUC (688h×ng/mL), whereas CPT319A and CPT307A increased AUC by 84 and 354 fold,respectively. Moreover, CPT319A exhibited a 5.5 h t_(1/2) compared tothe 1 h t_(1/2) of CPT307A, indicating that the cationic lipidDC-Cholesterol in CPT319A improves PK of the formulation by increasingcirculation time in the blood. The faster decrease of bloodconcentration for CPT319A compared to CPT307A in the first 3 h afterinjection indicates that the cationic charge of CPT319A causes a quickerdistribution to other organs, such as the liver.

TABLE 17 t_(1/2) and AUC values for Example 21. t_(1/2) Formulation (h)AUC (h × ng/mL) CPT319A 5.5 57453 CPT307A 1.0 243243 Naked DXR 688

Example 22 Programmable Liver Delivery of Liposome by Varying theCationic Lipid DC-Cholesterol Composition in the Liposome

In order to investigate the effects of cationic lipid DC-Cholesterol onin vivo distribution of liposomal DXR, the following three liposomeformulations were used for the PK studies: 1) Doxil® (FDA approvedliposomal DXR) containing no DC-Cholesterol, 2) CPT319A containing 15.3%DC-Cholesterol, and 3) CPT221 containing 38.3% DC-Cholesterol. The lipidcompositions of the three formulations are shown in Table 18.

Male CD-1 mice ranging from 20-25 g body weight were split up intogroups of three. Each mouse was administered with a single dose ofDoxil®, or CPT319A, or CPT221 at 5 mg/kg DXR by intravenous (IV)injection through the tail vein. The mice were sacrificed at designatedtimes and their livers were collected. For the groups treated withDoxil® and CPT221, livers were collected at 0.167, 0.5, 1, 2, 4, and 8 hafter the injection. For the group treated with CPT319A, livers werecollected at 0.167, 1, 3, and 8 h after the injection. The quantity ofDXR in the liver was determined using LC-MS-MS and the percentage of DXRin the liver was calculated against the total quantity of DXRadministered. The liver concentration curves of DXR are shown in FIG.14.

The cholesterol in Doxil® is completely substituted by DC-Cholesterol inCPT221, which resulted in a dramatic and rapid accumulation of DXR inthe liver. For example, 10 min after the injection, about 54% of DXR ofCPT221 was accumulated in the liver. The partial substitution ofcholesterol by DC-Cholesterol in CPT319A corresponded to a 21%accumulation of DXR in the liver, while only 9.4% DXR of Doxil®accumulated in the liver. Therefore, programmable liver-targetingdelivery can be achieved by controlling the molar ratio of a cationiclipid in the liposome.

TABLE 18 Lipid Compositions of Example 22. Component Doxil ® CPT319ACPT221 DOPC 61.7 DSPC 56.7 56.7 Cholesterol 38.3 17.7 DC-Cholesterol15.2 38.3 mPEG2000-DSPE 5.0 5.4 5.0 DXR 17.1* 10.3* 13.7* *this valuerepresents the molar % of DXR vs. total lipids.

1. A pharmaceutical composition comprising two types of liposomes: (i) afirst liposome type comprising a first lipid layer comprising anunsaturated phospholipid, cholesterol, and a first active pharmaceuticalingredient (API) comprising docetaxel in the first lipid layer; and (ii)a second liposome type comprising a second lipid layer, an aqueousinterior, and a second API comprising doxorubicin crystallized in theaqueous interior, wherein the first liposome type does not comprisedoxorubicin and the second liposome type does not comprise docetaxel. 2.The pharmaceutical composition of claim 1, wherein the first lipidlayer, the second lipid layer, or both the first and second lipid layersconsist essentially of the unsaturated phospholipid and cholesterol. 3.The pharmaceutical composition of claim 1, wherein the first lipidlayer, the second lipid layer, or both the first and second lipid layersconsist essentially of the unsaturated phospholipid, cholesterol, and apegylated phospholipid.
 4. The pharmaceutical composition of claim 1,wherein docetaxel is the only API in the first liposome type and/ordoxorubicin is the only API in the second liposome type.
 5. Thepharmaceutical composition of claim 1, wherein the first lipid layer,the second lipid layer, or both the first and second lipid layerscomprise: about 20-75% (molar) unsaturated phospholipid; about 10-60%(molar) cholesterol; and about 0-20% (molar) pegylated phospholipid. 6.The pharmaceutical composition of claim 1, wherein: the molar ratio ofthe first lipid layer components:docetaxel is about 100:1 to about 5:1;and the molar ratio of the second lipid layer components:doxorubicin isabout 100:1 to about 5:1.
 7. The pharmaceutical composition of claim 1,wherein the molar ratio of doxorubicin:docetaxel is about 10:1 to 1:10.8. The pharmaceutical composition of claim 1, wherein the unsaturatedphospholipid comprises a polyunsaturated phospholipid or amonounsaturated phospholipid.
 9. The pharmaceutical composition of claim1, wherein the cholesterol comprises a cationic cholesterol derivative.10. The pharmaceutical composition of claim 9, wherein the compositionis adapted to target one or more organs in a subject.
 11. Thepharmaceutical composition of claim 1, wherein the first liposomefurther comprises a pegylated phospholipid.
 12. (canceled)
 13. Thepharmaceutical composition of claim 1, formulated for intravenousadministration.
 14. The pharmaceutical composition of claim 1, whereinthe Z-average particle size of the liposomes is about 10-200 nm.
 15. Thepharmaceutical composition of claim 1, wherein, upon intravenousadministration to a subject, at least about 10% of the composition isdelivered to the liver.
 16. (canceled)
 17. (canceled)
 18. A method oftreating a subject comprising administering an effective amount of thepharmaceutical composition of claim 1 to the subject, wherein thesubject has a cancer.
 19. The method of claim 18, wherein the cancer isa lung cancer; colon cancer; breast cancer; stomach cancer; esophaguscancer; prostate cancer; leukemia; head and neck cancer; pancreaticcancer; multiple myeloma; or liver cancer.
 20. A method of making thepharmaceutical composition of claim 1, comprising: (i) making a firstliposome type by concurrently introducing a first lipid solution of anunsaturated phospholipid, a sterol, and docetaxel, in ethanol through afirst or plural inlet ports into a mixing chamber of a manifold andintroducing a first aqueous solution through a second or plural inletports into the mixing chamber of the manifold, and the liposomes exitthe mixing chamber through one or plural outlet chambers of themanifold, wherein the resulting first liposome type does not comprisedoxorubicin; (ii) making a second liposome type by concurrentlyintroducing a second lipid solution in ethanol through a first or pluralinlet ports into a mixing chamber of a manifold and introducing a firstaqueous solution through a second or plural inlet ports into the mixingchamber of the manifold, and the liposomes exit the mixing chamberthrough one or plural outlet chambers of the manifold; and incubatingthe resulting liposomes with doxorubicin, wherein the resulting secondliposome type does not comprise docetaxel; and (iii) combiningpredetermined amounts of the first liposome type and the second liposometype, thereby making the pharmaceutical composition of claim
 1. 21. Thepharmaceutical composition of claim 8, wherein the unsaturatedphospholipid comprises soy phosphatidylcholine or1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC).
 22. Thepharmaceutical composition of claim 9, wherein the cholesterol comprisesa dimethylaminoethanecarbamoyl-cholesterol (DC-cholesterol).
 23. Themethod of claim 20, wherein the first lipid solution further comprises apegylated phospholipid.